We are pursuing three complementary projects to elucidate the molecular pharmacology of clinically relevant inhibitors of topoisomerases and poly(ADPribose) polymerase (PARP) inhibitors. Project #1. Repair of topoisomerase cleavage complexes by tyrosyl-DNA-phosphodiesterases (TDPs) Aim 1: Biology of TDPs: TDP1 and TDP2 preferentially repair TOP1cc and TOP2cc, respectively. In addition to TOP1cc, TDP1 removes damaged and non-canonical bases and adducts from 3'-DNA ends. This explains why lack of TDP1 sensitizes cells not only to TOP1 inhibitors but also to temozolomide, cytarabine, zidovudine (AZT) and acyclovir. We are studying how TDP1 is regulated and recruited to DNA damaged sites. We recently reported that TDP1 is coupled with PARP1 and that inhibiting PARP1 results in TDP1 inactivation. Because TDP1 excises TOP1cc both in the nuclear and mitochondrial genomes, we are currently examining whether TDP2 also removes TOP2cc in the mitochondrial genome. We are also using knockout cell lines, site-directed mutagenesis and crystallography to elucidate the biology of TDPs. During this review period we showed that the two activities of TDP1, 3'- and 5'-phosphodiesterase depend on specific residues in the enzyme. We also showed for the first time that the arginine methyltransferase (PRMT5) activates TDP1 by directly binding and methylating TDP1. Aim 2: Pharmacology and targeting of TDPs: The rationale for targeting TDPs is rooted in the emerging importance of TDPs for DNA repair and viral replication, and the potential of TDP inhibitors for anticancer drug combinations. To do so, we are using biochemical assays with recombinant TDP enzymes. We are also taking advantage of TDP1 and TDP2 knockout cell lines, crystallographic determinations and molecular modeling to study the molecular pharmacology of the drug candidates. Project #2. PARP trapping by PARP inhibitors: molecular mechanisms and translational implications PARP inhibitors represent the most advanced cancer therapeutics targeting the DNA damage response, with one inhibitor, olaparib already an approved medicine and several others in late stage development. PARP inhibitors are the first drugs to exploit the concept of synthetic lethality for homologous recombination deficiency (HRD) in the clinic. Understanding the mechanism(s) of action of PARP inhibitors is key to the successful deployment of these agents. Our studies focus on 'PARP trapping' as an integral component of the pharmacology of these inhibitors. Aim 1: PARP pharmacology: Our studies focus on the differences in the molecular mechanisms of action between PARP inhibitors with respect to PARP trapping and what this means for both monotherapy activity and combination with chemotherapeutic agents. We are investigating in preclinical models the two most synergistic combinations: with temozolomide and with TOP1 inhibitors, including our non-camptothecin indenoisoquinoline TOP1 inhibitors (see above). Project #3. Genomic determinants of drug response and preclinical models for predictive biomarkers for patient selection and rational drug combinations with TOP1 and PARP inhibitors The insufficiencies of simple relationships between the activity of widely used DNA- and chromatin-targeted agents and their primary targets warrant the need to identify novel DNA damage response (DDR) determinants for predicting drug responses and rationalizing drug combinations. Aim 1: Use cancer cell line databases to mine drug responses based on CellMiner: Taking advantage of the extensive NCI-60 drug database ( 40,000 drugs including FDA approved and investigational clinical drugs), whole genomic data and our CellMiner facility, we discovered several novel predictive biomarkers for DNA-targeted agents: SLX4 (FANCP) mutations, ATAD5 (ELG1) mutations, and SLFN11 (Schlafen 11) expression. We are extending these analyses to tissue-specific cancer cell line databases (NCI Small Cell Lung Cancers), and larger databases (CCLE: MIT-BroadInstitute and CGP: MGH-Sanger), and to CCR clinical trials to test predictive biomarker signatures. Aim 2: SLFN11 as predictive biomarkers for response to DNA damaging drugs was discovered through NCI-60 analyses and in parallel by the CCLE teams. SLFN11 determines response to TOP1, TOP2, PARP inhibitors, DNA synthesis inhibitors and platinum derivatives but not to tubulin or protein kinase inhibitors or apoptosis-inducing drugs. SLFN11 is inactivated in approximately 50% of cancer cells lines, making them resistant to DNA damaging agents. Our aims are to elucidate the molecular mechanism of SLFN11 action and regulation, and relevance for patient responses and rationale drug combinations. We discovered a key molecular mechanism of action of SLFN11 by demonstrating that SLFN11 is recruited to DNA damage sites and to stressed replication forks by binding to RPA and the replicative CMG complex. In doing so, SLFN11 blocks elongating replicons and irreversibly blocks replication. We propose that SLFN11 acts as a Restriction Factor for cells with replicative stress.